STOMATAL RESPONSE TO WATER STRESS IN HERBACEOUS PERENNIALS

Introduction Herbaceous perennials planting have been popularly used in urban places, particularly in low water use landscaping because of their dynamic nature, low maintenance – cost saving and aesthetic appeal. To develop and maintain sustainable low water use landscape, we need to understand drought tolerance and drought responses of many ornamental plants, especially herbaceous perennials. During drought stress plants typically minimize water loss. Some methods of water conservation include decreases in stomatal conductance, differential growth of roots, shoots, and leaves, and changes in carbohydrate partitioning. Reduced stomatal conductance may lead to reduced photosynthesis (Prevete, Fernandez and Miller, 2000). Characterizing herbaceous perennials water deficit stress response mechanisms can inform selection of drought-tolerant herbaceous perennial species appropriate for urban landscape. Herbaceous perennials that avoid drought trough wilting, dormancy or dieback are less acceptable than species that tolerate drought by maintaining intact foliage (Zollinger et al., 2006). Some drought tolerance responses are better suited to urban landscapes. Deep-rooted, drought-avoiding species that become stressed and lose visual quality in shallow urban soils would be less suitable than species that withstand drought through stomatal closure and dehydration tolerance (Kjelgren, Wang and Joyce, 2009). Stomatal response to water stress was assessed for example for few ornamental herbaceous perennials: Echinacea purpurea, Gaillardia aristata, Lavandula angustifolia, Leucanthemum × superbum ‘Alaska’, Penstemon barbatus var. praecox nanus rondo, and Penstemon × mexicali ‘Red Rocks’ (Zollinger et al., 2006), Echinacea purpurea, Rudbeckia fulgida var. Sullivantii., Monarda didyma and Helianthus angustifolius (Chapman and Auge, 1994), Boltonia asteroides `Snowbank‘, Eupatorium rugosum and Rudbeckia triloba (Prevete, Fernandez and Miller, 2000). We sought to investigate drought responses of Stachys macrantha (C. Koch) Jalas and Brunnera macrophylla (Adams) IM Johnston., which are popular species of ornamental perennials grown commercially in nurseries.


Introduction
Herbaceous perennials planting have been popularly used in urban places, particularly in low water use landscaping because of their dynamic nature, low maintenance -cost saving and aesthetic appeal.To develop and maintain sustainable low water use landscape, we need to understand drought tolerance and drought responses of many ornamental plants, especially herbaceous perennials.During drought stress plants typically minimize water loss.Some methods of water conservation include decreases in stomatal conductance, differential growth of roots, shoots, and leaves, and changes in carbohydrate partitioning.Reduced stomatal conductance may lead to reduced photosynthesis (Prevete, Fernandez and Miller, 2000).
Characterizing herbaceous perennials water deficit stress response mechanisms can inform selection of drought-tolerant herbaceous perennial species appropriate for urban landscape.Herbaceous perennials that avoid drought trough wilting, dormancy or dieback are less acceptable than species that tolerate drought by maintaining intact foliage (Zollinger et al., 2006).Some drought tolerance responses are better suited to urban landscapes.Deep-rooted, drought-avoiding species that become stressed and lose visual quality in shallow urban soils would be less suitable than species that withstand drought through stomatal closure and dehydration tolerance (Kjelgren, Wang and Joyce, 2009).
We sought to investigate drought responses of Stachys macrantha (C.Koch) Jalas and Brunnera macrophylla (Adams) IM Johnston., which are popular species of ornamental perennials grown commercially in nurseries.

Experimental design
The study was conducted at the Experimental Station of Horticulture and Landscape Engineering Faculty in Nitra, over a 2-year period (2011)(2012).The two experiments were arranged as a split plot complete block design, with species and variant of experiment subplot.Each main plot consisted 1.5 l containers arranged in 3 rows with 15 replications.
The first experiment (two rows) was based on retaining stable drought level of soil water content: 30% and 60% soil water content.We retained stable drought level by adequate watering 3 times per week, without rainfall affect, because experiment was located in sheltered conditions (Clingfilm tunnel).The weight of the substrate determination at different soil water content at the beginning of the experiment was the key to watering amount.Each pot was regularly weight out before watering and adequate water content was replenished to the required level (30% and 60% soil water content).The second experiment (third row) was unirrigated, moisture in the soil drying up progressively from saturated soil condition to 30% soil water content.

Plant material
Stachys macrantha (C.Koch) Jalas and Brunnera macrophylla (Adams) IM Johnston.were planted in 1.5 l pots.Individual pots were filled with trade peat-clay medium Klassman TS-3.The weight of each planted pots and dry soil was determined for possibilities of calculation desired soil moisture content of trial pots.All plants were allowed to establish being kept well watered by saturating the growing media during the first month.

Drought treatments
Treatments of 30% and 60% soil water content (gravimetric measured as grams of water per gram of oven-dried soil) in trial pots were applied for two weeks in June 2011 and 2012.Necessary irrigation water was supplied to each trial pots according to foreordination, different in individual pots.

Data collection
Stomatal conductance, as an index of plant stress and indicator of photosynthetic activity, was measured with an AP4 Leaf Porometer.Measurements were taken on two fully expanded leaves of one plant with 10 replications per treatment between 7.00 and 15.00 hours.
Plants were harvested in mid-September.Leaves and stems were separated.All leaves per plant were detached to determine their relative water content (RWC).After cuttings, the petiole was immediately immersed in distilled water inside of a glass tube, which was immediately sealed.The tubes were then taken to the laboratory where the increased weight of the tubes was used to determine leaf fresh weight (FW).After 4 h, the leaves were weighed to obtain the turgid weight (TW).The dry weight (DW) was then measured after oven drying at 80 ºC for 48 h, and RWC was calculated as: RWC = 100(FW -DW)/TW -DW Leaf area was measured for green leaf tissue using the scanner, and then calculated by free software ImageJ.Specific leaf area (SLA) was calculated as the plant leaf area divided by the dry mass of leaves.Photosynthetic pigments like Leaf chlorophylls and carotenoids were extracted destructive method from fresh leaves with 85% acetone and estimated spectrophotometrically as describe by Šesták and Čadský (1966).

Statistical analysis
Statistical analyses of experimental data were performed using Statgraphics Plus 4.0® (Statistical Grafics Corp., Herndon, Va.U.S.A.).Analysis of variance (ANOVA) was performed to estimate statistically significant differences between their averaged values at a confidence level of 95% (P-value <0.05).A multiple range test of least significant difference test (LSD tests) was used to analyze the existence of homogenous samples.

Results and discussion
The present study was aimed at better understanding the physiological and morphological response of this species to drought stress, so that to their recommendation to low water use landscaping.Brunnera macrophylla showed great tolerance to levels of drought, avoiding desiccation by decreasing stomatal conductance as water became limiting (Zollinger et al., 2006).Compared to control plants of (60% soil water content) stomatal conductance was reduced by 67.7% and 65.1% (table 1) during two observation years.We got the same results with Stachys macrantha, when stomatal conductance was reduced by 48% and 76.4% (only one year significantly) (table 1).
Stomatal conductance linearly decreased during the drying cycle in Brunnera macrophylla (table 2, Figure 1) and Stachys macrantha (table 3, Figure 2).Stomatal conductance of Brunnera macrophylla fell most rapidly.Traditionally, leaf water potential has been considered to be the primary parameter controlling stomatal behavior during drought, but others have indicated that stomatal closure was better correlated with leaf turgor potential, recent investigations suggest that stomatal closure is directly linked to soil drying (Chapman and Auge, 1994).
All species were poorly morphological response to the drought stress.We observed significant differences between drought treatment (table 4) on Brunnera macrophylla and Stachys macrantha only in leaf morphology (leaf area and leaf dry matter content).
Specific leaf area (SLA), an indicator of leaf thickness, has often been observed to be reduced under drought conditions (Marcelis, Heuvelink and Goudriaan, 1998).Low SLA is preferable as it indicates higher drought resistance (Painawadee et al., 2009).A decrease in SLA may also occur in response to drought in herbaceous leaves as a result on an increased investment in structural tissues, allowing increased resistance to unfavorable environmental conditions (Maroco, Pereira and Chaves, 2000).It could by hypothesized that all investigated species with low SLA have more photosynthetic machinery per unit leaf area and hence potential for greater assimilation under drought stress because thicker leaves usually have a greater photosynthetic capacity compared with thinner leaves (Painawadee et al., 2009).Investigated plants show poorly, mainly non significant decreased SLA (Brunnera macrophylla by 5% and 14.7%*, and Stachys macrantha by 2.2%, and non significant increased by 0.3% in 2012).The consistency of SLA makes this parameter useful for use as a selection criterion in drought resistance plants for low water use landscaping.
The leaf area was most affected at the drought stress for all species.Differences between the control (60%   (Liu and Stutzel, 2004).Stomatal acclimation would by more ornamentally desirable, than the plants acclimate by reducing total transpiration via drastically eliminating leaf area (Zollinger et al., 2006), especially for ornamental plantings.Our conclusion based on this consideration is that Brunnera maccrophylla can withstand better moderate water losses without aesthetic deprivation than Stachys macrantha.
Drought stress increase leaf dry matter and sugar content (Schut And Ketelaars, 2003).All drought treated plants in our study had higher dry weights of than controls, in case Stachys macrantha and Brunnera macrophylla significantly.Leaf dry matter content significantly increased by 18.5% and 16.7% (Brunnera macrophylla) and by 12% (Stachys macrantha).
Relative water content (RWC) compares the water content of a leaf with the maximum water content at full turgor.RWC, as a stress indices allow consideration the quantity of water in the plants (Ceccato et al., 2001), reflected the metabolic activity in tissues and used as a most meaningful index for dehydration tolerance (Anjum et al., 2011).RWC of leaves is higher in the  The finally study aim was investigation of physiological response on drought treatment by evaluation the levels of chlorophylls and carotenoids (Table 5).Chlorophyll and carotenoid absorb radiant energy, which is used for photosynthesis.In many observed cases chlorophyll content declines under stress conditions (Nazarli, Faraji and Zardashti, 2011).
Chlorophyll loss is a negative consequence of stress, on the other hand, it has also been considered as an adaptive feature in plants grown under extreme climatic conditions.Chlorophyll loss may also contribute to the survival of severely stressed plants by reducing the amount of photons absorbed by leaves, which leads to an enhanced photoprotective and antioxidant capacity of leaves per amount photons absorbed (Munné-Bosch and Alegre, 2000).The significant differences in leaf chlorophyll (Chl) contents were detected between drought stressed and control plants (Table 5).Drought stress decreased total chlorophyll content by 18.2%* and 9.7% (Brunnera macrophylla) and by 11.9% and 20.6%* (Stachys macrantha).
Carotenoids show multifarious roles in drought tolerance including light harvesting and protection from oxidative damage caused by drought.Thus, increased contents specifically of carotenoids are important for stress tolerance (Painawadee et al., 2009).The carotene content was found to increase (table 5) with drought treatment of Brunnera macrophylla (by 22.1%*) but contrariwise results was found with drought treatment of Stachys macrantha, when carotene content was decrease (by 19.8%*).

Figure 1
Figure 1Simple correlation among % soil water content and stomatal conductance of Brunnera macrophylla

Table 1
Stomatal conductance (g s ) in mm s -1 across species and drought treatments Values are ± standard error.Different letter indicate significant differences in stomatal conductance (P ≤0.05) between drought treatments for each species Dagmar Hillová, Magdaléna Takácsová, Helena Lichtnerová: Stomatal response to water stress in herbaceous perennials, pp.52-56

Table 2
Simple correlation among % soil water content and stomatal conductance of Brunnera macrophylla

Table 3
Simple correlation among % soil water content and stomatal conductance of Stachys macrantha Simple correlation among % soil water content and stomatal conductance of Stachys macrantha

Table 4
Leaf areas, Specific leaf area, Relative Water content (RWC), Leaf dry matter content across species and drought treatments

Table 5
Total chlorophyll and total carotenoid across species and drought treatments

Species Observation year Drought treatment (% soil water content) Total Chl in mg m -2 leaf area Total carotenoid in mg m -2 leaf area
(Gong et al., 2010))88055212623.52-56Plants in Urban Areas and Landscape Slovak University of Agriculture in Nitra Faculty of Horticulture and Landscape Engineering initial stages of leaf development and declines as the dry matter accumulates and leaf matures.RWC related to water uptake by the roots as well as water loss by transpiration(Anjum et al., 2011).Drying soil led to continuous decrease in RWC of leaf, but decreasing rate varied with different species.There was no significant difference in leaf RWC for Brunnera macrophylla and Stachys macrantha.The drought-tolerant plants, with higher RWC displayed superior water maintenance and hydraulic conductance abilities to drought-sensitive one(Gong et al., 2010).The result show, that Brunnera macrophylla and Stachys macrantha can withstand moderate water losses, because RWC remained the same in drought treatment.
Values are ± standard error.Different letter indicate significant differences in totalchlorophyll and total carotenoid content (P ≤0.05) between drought treatments for each species Dagmar Hillová, Magdaléna Takácsová, Helena Lichtnerová: Stomatal response to water stress in herbaceous perennials, pp.52-56